Method for influencing the p53 linkage to target genes

ABSTRACT

The invention relates to a method for influencing the p53 binding to a target gene, wherein the conformations of p53 and the target gene are coordinated especially by means of conformation modulators and the binding of p53 can be directly or indirectly detected.

This is a national phase filing of the Application No. PCT/DE98/02326,which was filed with the Patent Corporation Treaty on Aug. 7, 1998, andis entitled to priority of the German Patent Application 197 35 221.9,filed Aug. 14, 1997.

I. FIELD OF THE INVENTION

The present invention relates to a method for influencing the p53binding to target genes.

II. BACKGROUND OF THE INVENTION

p53 is a protein present in animal and human cells and is referred to asa “guardian of the genome.” p53 is a sequence-specific transactivatorwhich is activated in the case of DNA damage. In this form, p53 binds topromoters of target genes and activates the transcription thereof. Thiscauses growth stand-still of the cells and subsequent repair of the DNAdamage and cell death, respectively.

It has turned out that p53 has lost its transactivator activity in manytumors. This is often due to the fact that the binding of p53 to thepromoters of target genes is disturbed or it is not the desired targetgenes that are regulated.

Therefore, it is the object of the present invention to provide aproduct by which it is possible to influence the binding of p53 totarget genes.

III. SUMMARY OF THE INVENTION

The invention relates to a method for influencing the p53 binding to atarget gene, wherein the conformations of p53 and the target gene arecoordinated especially by means of conformation modulators and thebinding of p53 can be directly or indirectly detected.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA conformations of oligonucleotides (SEQ ID NOS: 1-3)which contain on their termini the p53 binding sequence RGC. SEQ ID NO:2 is in conformation II (i.e., B DNA conformation), while SEQ ID NOS:1and 3 are in conformation I and III (i.e., non-B DNA conformation). Todemonstrate the different conformations, RGC containing oligonucleotidesare subjected to an annealing reaction with an oligonucleotide,designated “Lock,” (SEQ ID NO:4) that has complementary sequences to thesequence of the RCG sequences on the termini of SEQ ID NOS:1-3. Theautoradiograph of a polyacrylamid gel is shown on which the the variousreaction products has been separated. Lanes 2, 4, and 6 show theannealing product of SEQ ID NOS:1, 2, and 3, respectively, with “Lock”(SEQ ID NO:4) where “Lock” was radioactively labeled. Lane 1 showsradioactively labeled “Lock” as control. Lane 8 shows radioactivelylabeled RCG containing oligonucleotides as control.

FIG. 2 shows the DNA binding of p53 to RCG containing oligonucleotides(SEQ ID NOS:1, 2, and 10). Lanes 3 and 4 show binding to RCG 1a/ss (SEQID NO:1), lanes 5 and 6 show the binding to RCG 1b/ss (SEQ ID NO:10),and lanes 7 and 8 show the binding to RCG dm/ss (SEQ ID NO:2). Lanes 1and 2 represent controls.

FIG. 3 shows the DNA conformations of oligonucleotides which contain p53binding sequences of the sequence found by Hupp (SEQ ID NOS:5-6)annealed to Lock-2 (SEQ ID NO:8), and (SEQ ID NO:7) annealed to (SEQ IDNO:9).

FIG. 4 shows the DNA binding of p53 to oligonucleotides by p53 bindingsequences of the Hupp sequences (SEQ ID NOS:5-7), the oligonucleotideshaving various DNA conformations. (A) indicates the absence, (B) thepresence of antibody pAb421.

V. DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a product by whichit is possible to influence the binding of p53 to target genes.

According to the invention this is achieved by the subject mattersdefined in the claims.

Thus, the subject matter of the present invention relates to a method ofinfluencing the binding of p53 to a target gene in which theconformations of p53 and the target gene are coordinated, particularlyby means of conformation modulators, and the binding of p53 is detecteddirectly or indirectly.

The present invention is based on the applicant's insight that p53binding sequences in the promoters of target genes can be present notonly in the “ordinary” B-duplex DNA conformation but also in non-B DNAconformations (e.g., cruciate). The applicant also discovered that p53identifies its binding sequences in both the B-duplex DNA conformationand in the non-B DNA conformations. However, the identification differsdepending on the conformation of p53.

According to the invention these insights are used to influence tobinding of p53 to target genes by coordinating the conformations of p53and the target genes, particularly by means of conformation modulators,and detecting the p53 binding directly or indirectly.

The expression “p53” comprises a p53 of any kind and origin. It can havea wild-type sequence or be a mutated p53. The latter is preferably a p53with mutated ability of DNA binding. p53 can also be present in the formof a fragment of p53 which is responsible for the DNA binding. Inaddition, p53 can be present in the form of a fusion polypeptide. Likeany other p53 it can also be present in the form of a vector coding forit.

The term “target genes” comprises genes of any kind and origin theexpression of which is regulated by p53. Examples of such genes are RGC,MCK, mdm2, cyclin G, synthetic p53 reporter genes, p21 and bax, p21 andparticularly bax being preferred. p21 is held responsible for the growthstand-still of the cell caused by p53 and bax is held responsible forthe cell death caused by p53. In particular, the expression “targetgenes” comprises the promoter sequences thereof and more particularlyp53 binding sequences thereof. The target genes may be present in anyDNA conformation. They can be present in cells, particularly tumorcells, or occur in isolated fashion. The target genes can also bepresent in connection with further sequences, particularly with thosecoding for a reporter protein.

The expression “conformation modulators” comprises substances of anykind which can cause a conformational change of a nucleic acid. Inparticular, substances are concerned which can convert a DNA from theB-duplex DNA conformation into a non-B DNA conformation or vice versa.Examples of such substances are intercalating substances. The expression“conformation modulators” also comprises substances of any kind whichcan effect a conformational change of p53. Examples of such substancesare those which can modify the carboxy-terminal regulator region of p53,e.g., antibody pAB 421.

The expression “direct or indirect detection of p53 binding” comprisesany detection for such a binding. Examples of a direct detectioncomprise methods by means of which the p53 binding to DNA can be shown.Indirect detections include methods by means of which it is possible toshow the consequences of a p53 DNA binding, e.g., the regulation of theexpression of reporter genes and/or biological consequences, such as thegrowth stand-still of cells or the death thereof.

A method according to the invention can be carried out as usual. Forexample, p53 binding sequences in the B-duplex DNA conformation and in anon-B DNA conformation, respectively, can be incubated with p53 andoptionally conformation modulators and the DNA binding of p53 isdetected directly. It is possible to incubate cells, particularly tumorcells, having disturbed DNA binding of p53 with conformation modulatorsand upon irradiation of the cells the DNA binding of p53 is detectedindirectly via the growth stand-still and the death of the cells,respectively.

By means of the present invention it is possible to influence the p53binding to target genes. This can be used to correct a disturbed DNAbinding of p53, which often exists in tumor cells. Furthermore, the DNAbinding specificity of p53 can be influenced so as to regulate orcontrol certain desired target genes. Thus, the present invention can beused at least as an accompanying treatment measure to combat tumoraldiseases.

The present invention also offers the possibility of discoveringsubstances which can be suitable as conformation modulators of p53and/or its target genes. For this purpose, the method according to theinvention is carried out to the effect that in place of knownconformation modulators unknown ones are used and the known conformationmodulators are optionally used as controls.

The below examples explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

VI. EXAMPLES A. Example 1 p53 Binding To A Target Gene

(a) p53 Binding to p53 Binding Sequences of RGC

p53 was isolated as usual from Sf9 insect cells which were infected withrecombinant wild type p53-expressing baculoviruses. p53 had a purity ofover 80% in an SDS polyacrylamide gel. p53 was used in a bindingreaction with oligonucleotides. For preparing these oligonucleotides,the below oligonucleotides were used as a basis:

Hu/La-1

(SEQ ID NO:6) Hu/La-2

(SEQ ID NO:5) Hu/La-du

SEQ ID NO:7)

These oligonucleotides contain 1-2 “halfsites” of the p53 bindingsequences of RGC and in each case identical terminal sequences at their5′ ends and 3′ ends. The oligonucleotides were used in an “annealing”reaction with the below oligonucleotide which has complementarysequences with respect to the terminal sequences:

Lock 5′-ccgcggtaccattacctaaggcgtc-3′  (SEQ ID NO:4)

Oligonucleotides were obtained which are present in the duplex-B DNAconformation (structure II) and in the non-B DNA conformations,respectively (structures I and III, respectively). See, FIG. 1. Theseoligonucleotides were terminally labeled radioactively and used in thebinding reaction with p53. The reaction lasted 30 minutes at roomtemperature. The reaction products were subjected to polyacrylamide gelelectrophoresis.

It turned out that p53 binds to the oligonucleotides of the three DNAconformations, although to differing degrees. The strongest bond isfound in the case of a non-B DNA conformation (structure I), while thebond to the duplex-B DNA conformation (structure II) and to anothernon-B DNA conformation (structure III), respectively, is weaker. See,FIG. 2. Thus, it becomes evident that the p53 binding to a target genecan be influenced by a conformational change of the target gene.

(b) p53 Binding to p53 Binding Sequences of the Hupp Sequence

In accordance with the description of (a), p53 was used in a bindingreaction with oligonucleotides. For the preparation thereof, the belowoligonucleotides were used as a basis:

RGC - 1 b/ss 5′-gacgccttaggtagggccctGGACTTGCCTcccgggatggtaccgcgg-3′ (SEQID NO:10) RGC - 1 a/ss

(SEQ ID NO:1) RGC-dm/ss

(SEQ ID NO:2)

They contain 1-2 “halfsites” of the p53 binding sequences discovered byHupp. These p53 binding sequences distinguish themselves in that p53 canbind thereto only after its activation by the antibody PAb 421. Theoligonucleotides were used in an “annealing” reaction with theoligonucleotide “lock” indicated in (a). (SEQ ID NO:4). Oligonucleotideswere obtained which are present in the duplex-B DNA conformation(Hu/La-du/ds) (SEQ ID NO:7) and in the non-B DNA conformations,respectively (Hu/La-2 (SEQ ID NO:5) and Hu/La-1 (SEQ ID NO:6),respectively). See, FIG. 3.

It turned out that p53 binds to the oligonucleotides of the three DNAconformations, although to differing degrees. p53 binds to theoligonucleotide in the duplex-B DNA conformation only after theactivation of p53 by the PAb 421 antibody, while its binding to non-BDNA conformations takes place without modification of p53. See, FIG. 4.On the other hand, the binding of PAb 421 to p53 effects an inhibitionof the p53 binding to this oligonucleotide in the non-B DNAconformation. Hence it becomes evident that the p53 binding to a targetgene can be influenced by conformational changes of the target gene andof p53.

10 1 58 DNA Artificial Sequence Description of Artificial Sequence p53binding oligonucleotide RGC-1a/ss 1 gacgccttag gtacctggcc tgcctggacttgcctggcct gcctggatgg taccgcgg 58 2 67 DNA Artificial SequenceDescription of Artificial Sequence p53 binding oligonucleotide RGC-dm/ss2 gacgccttag gtacctgcct ggacttgcct ggtcctccag gcaagtccag gcaggatggt 60accgcgg 67 3 58 DNA Artificial Sequence Description of ArtificialSequence p53 binding oligonucleotide RGC in conformation III 3gacgccttag gtacctggac ttgcctggcc tgcctggact tgcctatggt accggcgg 58 4 25DNA Artificial Sequence Description of Artificial Sequence “Lock”annealing oligonucleotide 4 ccgcggtacc attacctaag gcgtc 25 5 32 DNAArtificial Sequence Description of Artificial Sequence Bindingoligonucleotide Hu/La-2 5 gccgccagct tagacatgcc tatcgaccgc cg 32 6 42DNA Artificial Sequence Description of Artificial SequenceBindingoligonucleotide Hu/La-1 6 gccgccagct tagacatgcc tagacatgcc tatcgaccgc cg42 7 26 DNA Artificial Sequence Description of ArtificialSequenceBinding oligonucleotide Ha/La-du 7 agcttagaca tgcctagaca tgccta26 8 20 DNA Artificial Sequence Description of Artificial Sequence“Lock-2” annealing oligonucleotide 8 cggcggtcga agctggcggc 20 9 25 DNAArtificial Sequence Description of Artificial Sequence annealingoligonucleotide 9 tcgaatctgt acggatctgt acgga 25 10 48 DNA ArtificialSequence Description of Artificial Sequence p53 binding oligonucleotideRGC-1b/ss 10 gacgccttag gtagggccct ggacttgcct cccgggatgg taccgcgg 48

What is claimed:
 1. A method for identifying a conformation modulator ofa p53 target nucleic acid sequence, comprising: (a) providing a p53molecule and a p53 target nucleic acid sequence; (b) exposing said p53molecule and said p53 target nucleic acid sequence to a candidatesubstance; and (c) identifying that said candidate substance is aconformation modulator of a p53 target nucleic acid sequence bycomparing binding of said p53 molecule to said p53 target nucleic acidsequence in the presence of said candidate substance to binding of saidp53 molecule to said p53 target nucleic acid sequence in the absence ofsaid candidate substance.
 2. The method of claim 1, wherein saidconformation modulator is a substance that converts the p53 targetnucleic acid sequence from B-duplex formation to a non B-duplexformation.
 3. The method of claim 1, wherein said conformation modulatoris an intercalating substance.
 4. The method of claim 1, wherein saidp53 target nucleic acid sequence is p21 or bax.
 5. The method of claim1, wherein said p53 molecule is a truncated form of p53, said truncatedform of p53 having the ability to bind a p53 target nucleic acidsequence.
 6. The method of claim 1, wherein said p53 molecule is afusion polypeptide.
 7. The method of claim 1, wherein binding of saidp53 molecule to said p53 target nucleic acid sequence is detecteddirectly.
 8. The method of claim 1, wherein binding of said p53 moleculeto said p53 target nucleic acid sequence is detected indirectly.
 9. Themethod of claim 1, 2, or 3, wherein said p53 is a mutated p53.
 10. Themethod of claim 1, 2, or 3, wherein said p53 molecule is a wild-type p53molecule.
 11. The method of claim 1, 2, or 3, wherein said p53 targetnucleic acid sequence regulates the expression of a synthetic reportergene.
 12. The method of claim 9, wherein said mutation of p53 affectsthe DNA binding affinity of said mutated p53.
 13. The method of claim 9,wherein binding of said p53 molecule to said p53 target nucleic acidsequence is detected by the expression of a reporter gene, wherein saidp53 target nucleic acid sequence regulates the expression of saidreporter gene.
 14. The method of claim 8, wherein binding of said p53molecule to said p53 target nucleic acid sequence is detected by thebiological consequence of p53 binding to said p53 target nucleic acidsequence, wherein said biological consequence is cell growth arrest. 15.The method of claim 1, wherein binding of said p53 molecule to said p53target nucleic acid sequence is detected by the biological consequenceof p53 binding to said p53 target nucleic acid sequence, wherein saidbiological consequence is cell death.